Protein-nucleic acid complexes are known to play an important role in a variety of biological processes. See, e.g., Hill et al. "Fluorescence Approaches to Study of Protein-Nucleic Acid Complexation," 278 Methods in Enzymology 390 (1997). For example, DNA-binding proteins are known to play an important role in gene regulation. Genes are typically regulated at the transcriptional level by DNA-binding proteins, which are referred to as transcription factors. Transcription factors regulate gene expression by specifically binding to a target nucleic acid sequence in promoter DNA.
Due to the biological importance of protein-nucleic acid interaction, a variety of methods for studying protein-nucleic acid binding characteristics have been proposed. See, e.g., Hill et al. and the references cited therein.
U.S. Pat. No. 5,783,384 to Verdine discloses methods for determining the affinity of a DNA-binding protein for a target nucleic acid sequence. Verdine teaches methods comprising providing a reversible bond between a DNA-binding protein and a target nucleic acid sequence, and determining the relative strength of the reversible bond (and thus the affinity of the protein for the nucleic acid) by breaking it under supervised conditions. The more stringent the conditions necessary to break the bond, the higher the affinity of the protein for the nucleic acid. Verdine does not disclose or suggest fluorescence-based binding assays.
U.S. Pat. No. 5,445,935 to Royer discloses fluorescence-based methods for studying protein-oligonucleotide binding; however, the teachings of the patent are solely limited to fluorescent anisotropy techniques. Basically, anisotropy measures rotational diffusion events of free DNA or protein-bound DNA, as well as the local motions of a fluorophore attached to the DNA via a linker arm. Free DNA rotates quickly, depolarizes the light more readily and exhibits a low anisotropy value. In contrast, protein-bound DNA rotates slowly relative to the lifetime of the fluorophore, depolarizing the light only slightly and thus exhibiting a relatively high anisotropy value.
However, there are significant drawbacks to anisotropy-based assays. The degree of change in anisotropy as a function of binding is not as predictable as the proponents of anisotropy-based methods assert. Interpretation of anisotropy data to conform inconsistent data to theoretical expectations can require more effort than is desirable in an analytical method, particularly when the method is to be automated.
Radioactive labeling remains the most popular method for analyzing protein-nucleic acid interaction, despite being relatively slow, a health and environmental hazard, and relatively labor-intensive. Conventional radioactive labeling methods typically require radioactively end-labeling DNA probes with .sup.32 P using specialized enzymes. Purification of labeled DNA from unincorporated .sup.32 P involves polyacrylamide gel electrophoresis, overnight elution, gel filtration and concentration steps. Since the half-life of .sup.32 P is only 14 days, radio-labeling is required approximately every three weeks for each probe. Moreover, protein-.sup.32 P-DNA complexes need to be separated from unbound .sup.32 P-DNA by native polyacrylamide gel electrophoresis. Gels are then dried and analyzed by autoradiography or phosphoimaging.
Thus, a need has existed in the art for a simple, effective and rapid method for analyzing peptide-nucleic acid and protein-nucleic acid interaction.
All references cited herein, including U.S. Pat. No. 5,846,729 and U.S. patent applications Ser. Nos. 08/807,901 and 08/870,370 (respectively filed Feb. 27, 1997 and Jun. 6, 1997), are incorporated herein by reference in their entireties.